Immunochemical characterization of the pyruvate dehydrogenase complex in adult Ascaris suum and its developing larvae

Abstract

Polyclonal antibody was prepared against the pyruvate dehydrogenase complex purified from adult Ascaris suum body wall muscle. The antibody reacted with the E2, X, αE1 and βE1 subunits of the complex in immunoblots of mitochondrial supernatant fractions and homogenates of adult muscle. In addition, the same subunits were observed in immunoblots of homogenates of L3 and L4 ascarid larvae, suggesting that a similar enzyme complex was present in all developmental stages despite their marked differences in energy metabolism. The phosphorylated and dephosphorylated αE1 peptides migrated differently during sodium dodecylsulfate polyacrylamide gel electrophoresis and both forms of the enzyme were recognized by the antibody. These results and those obtained with ELISA suggest that both phosphorylated and dephosphorylated forms of the αE1 subunit react equally well with the antibody. In immunoblots of adult body wall muscle, the phosphorylated αE1 peptide predominated, while immunoblots of L3 larvae contained predominantly the dephosphorylated form. These results reflect the in vivo activity state of the pyruvate dehydrogenase complex in these two stages and suggest that this technique may be useful for determining the activity state of enzyme complex directly from immunoblots of homogenates A. suum and other helminths.